Assay modules having assay reagents and methods of making and using same

ABSTRACT

We describe assay modules (e.g., assay plates, cartridges, multi-well assay plates, reaction vessels, etc.), processes for their preparation, and method of their use for conducting assays. Reagents may be present in free form or supported on solid phases including the surfaces of compartments (e.g., chambers, channels, flow cells, wells, etc.) in the assay modules or the surface of colloids, beads, or other particulate supports. In particular, dry reagents can be incorporated into the compartments of these assay modules and reconstituted prior to their use in accordance with the assay methods. A desiccant material may be used to maintain and stabilize these reagents in a dry state.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/752,475, filed Dec. 21, 2005; U.S. Provisional Application No.60/752,513, filed Dec. 21, 2005; and U.S. application Ser. No. 11/______(Atty. Dkt. 4504-16), filed Dec. 21, 2006, entitled “Assay Apparatuses,Methods and Reagents”; each of which is hereby incorporated byreference.

STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

This invention was made with federal support under HDTRA1-05-C-0005awarded by Department of Defense. The U.S. government has certain rightsin the invention.

FIELD OF THE INVENTION

The invention relates to assay modules, such as assay plates,cartridges, multi-well assay plates, reaction vessels, and methods forconducting chemical, biochemical, and/or biological assays. Theinvention also relates to the incorporation of dry reagents into thesemodules and/or the use dry reagent in these methods.

BACKGROUND OF THE INVENTION

Numerous methods and systems have been developed for conductingchemical, biochemical, and/or biological assays. These methods andsystems are essential in a variety of applications including medicaldiagnostics, food and beverage testing, environmental monitoring,manufacturing quality control, drug discovery, and basic scientificresearch.

Depending on the application, it is desirable that assay methods andsystems have one or more of the following characteristics: i) highthroughput, ii) high sensitivity, iii) large dynamic range, iv) highprecision and/or accuracy, v) low cost, vi) low consumption of reagents,vii) compatibility with existing instrumentation for sample handling andprocessing, viii) short time to result, ix) multiplexing capability, andx) insensitivity to interferents and complex sample matrices. It is alsodesirable in many applications that these types of performance benefitsare achieved with assay formats that are easy to carry out, are amenableto automation, and/or use stable dry reagents. There is substantialvalue to new assay methods and systems with these characteristics.

A variety of approaches have been developed that provide reagents forassays in dry stable form. U.S. Pat. No. 5,413,732 describes certain dryreagent spheres that are capable of dissolving in a solution.

U.S. Pat. No. 6,429,026 describes certain immunoassays using dryreagents and time-resolved fluorescence detection. A catching antibodyis immobilized on the surface of a microtitration well. An insulatinglayer containing carbohydrate and/or protein is dried on top of thecatching antibody at the bottom of the well. A labeled antibody is addedin a small volume and dried on top of the insulating layer. The antibodyis labeled with a lanthanide chelate that can be detected usingdissociation enhance lanthanide fluoroimmunoassay (DELFIA) techniques.To start the immunoassay, a sample and a common assay buffer is added.After allowing the antibody reactions to occur, the well is washedseveral times, a DELFIA enhancement buffer is added, and a fluorescencelifetime measurement is carried out.

U.S. Publication 2003/0108973 describes a sandwich immunoassay thatemployed a test tube containing a lyophilized mixture comprising acapture antibody immobilized on 2.8 μm magnetizable polystyrene beadsand a detection antibody labeled with an electrochemiluminescent label.The mixture could also include blocking agents to reduce non-specificbinding of the detection antibody to the beads during the lyophilizationprocess. Addition of sample containing the analyte of interest resultedin the formation of sandwich complexes on the beads. A suspension ofbeads was then aspirated into a reusable flow cell where they werecollected on an electrode and analyzed using electrochemiluminescence(ECL) detection techniques.

U.S. Pat. No. 6,673,533 of Wohlstadter et al. describes an ECL-basedsandwich immunoassay using dry reagents. A capture antibody wasimmobilized on a composite electrode. The other reagents used in assaywere dried on the electrode surface by adding and lyophilizing asolution containing a detection antibody linked to an ECL label,phosphate, tripropylamine, bovine serum albumin, sucrose,chloracetamide, and TRITON X-100. Immunoassays were conducted by addinga sample to the dried reagents on the electrodes, incubating thesolutions, and applying a potential to the electrode to induce ECL. Nowashing step was required.

A variety of techniques have been developed for increasing assaythroughput. The use of multi-well assay plates (also known as microtiterplates or microplates) allows for the parallel processing and analysisof multiple samples distributed in multiple wells of a plate. Multi-wellassay plates can take a variety of forms, sizes, and shapes. Forconvenience, some standards have appeared for instrumentation used toprocess samples for high-throughput assays. Multi-well assay platestypically are made in standard sizes and shapes, and have standardarrangements of wells. Arrangements of wells include those found in96-well plates (12×8 array of wells), 384-well plates (24×16 array ofwells), and 1536-well plates (48×32 array of wells). The Society forBiomolecular Screening has published recommended microplatespecifications for a variety of plate formats (seehttp://www.sbsonline.org).

U.S. Publications 2004/0022677 and 2005/0052646 of U.S. application Ser.Nos. 10/185,274 and 10/185,363, respectively, of Wohlstadter et al.describe solutions that are useful for carrying out singleplex andmultiplex ECL assays in a multi-well plate format. They include platesthat comprise a plate top with through-holes that form the walls of thewells and a plate bottom sealed against the plate top to form the bottomof the wells. The plate bottom has patterned conductive layers thatprovide the wells with electrode surfaces that act as both solid-phasesupports for binding reactions as well as electrodes for inducing ECL.The conductive layers may also include electrical contacts for applyingelectrical energy to the electrode surfaces.

Despite such known methods and systems for conducting assays, improvedassay modules for conducting chemical, biochemical, and/or biologicalassays are needed.

SUMMARY OF THE INVENTION

The invention relates to assay modules (e.g., assay plates, cartridges,or multi-well assay plates, reaction vessels, etc.), having assayreagents pre-loaded in the wells, chambers or assay regions of the assaymodule. In certain embodiments, these assay reagents are stored in a drystate. Furthermore, the assay modules may comprise desiccant materialsfor maintaining these assay reagents in a stable dry state. A method isprovided for making such assay modules and methods for using the assaymodules in assays.

A multi-well plate is provided comprising at least one well having (1) abinding surface having a first binding reagent immobilized thereon and(2) at least one additional dry reagent, wherein at least one additionaldry reagent does not contact the binding surface. The multi-well platemay have an electrode surface with a binding surface incorporated in atleast one well of the multi-well plate.

A multi-well assay plate is provided comprising a plate body with aplurality of wells defined therein, the plurality of wells comprising abinding surface having a capture reagent immobilized thereon and areconstitutable dry reagent. Optionally, the binding surface may beselected to be suitable for use as an electrode in an electrochemicalassay or electrochemiluminescence assay. Furthermore, the bindingsurfaces may be coated with a reconstitutable protective layer. The dryreagent, which may be a labeled detection reagent, is free standing orlocated on a surface of the well that does not overlap with the bindingsurface. In one specific example, the binding surface is located on thebottom of the well and the reconstitutable dry reagent is located on awall of the well and, optionally, on a reagent storage shelf defined onthe wall. In another example, the binding surface and thereconstitutable dry reagent are both located on non-overlapping regionsof the bottom surface of the well. In another specific example, thereconstitutable dry reagent is a free-standing pill.

The multi-well assay plate may further comprise a reconstitutable dryassay control analyte which may have binding affinity for theimmobilized capture reagent and/or, if present, the labeled detectionreagent. In certain embodiments, the control analyte has affinity forimmobilized capture reagents and/or labeled detection reagents withinthe well, but is present in unbound form that is not in contact with thebinding surface or labeled detection reagent.

The multi-well assay plate may further comprise one or more additionalimmobilized capture reagents. The capture reagent and additional capturereagents are patterned on the binding surface to form an array ofbinding domains on the binding surface. These binding domains/capturereagents may differ in specificity or affinity for binding partners. Inaddition, the wells may contain a plurality of different reconstitutabledry labeled detection reagents that differ in specificity or affinityfor binding partners.

The multi-well plates, described above, may be used in methods ofcarrying out assays comprising adding sample to one or more of the wellsof a plate comprising immobilized capture reagents and reconstitutabledry labeled detection reagents, reconstituting reconstitutable drymaterials in these wells to form a reaction mixture(s), incubating thereaction mixture(s) under conditions that promote binding of saidcapture and detection reagents to their corresponding binding partners,and measuring the formation of complexes comprising the immobilizedcapture reagents and labeled binding reagents. By appropriate choice ofcapture and detection reagents, these methods may include sandwichbinding assay methods and competitive binding assay methods.

A method is provided of preparing multi-well assay plates for use in anassay comprising carrying out the following on at least two wells of aplate: immobilizing a capture reagent on a surface of a well of saidplate to form a binding surface, dispensing a liquid reagent comprisinga labeled detection reagent to a surface of the well that does notoverlap the binding surface, and drying the liquid reagent to form areconstitutable dry detection reagent. The method may also include,dispensing a protecting reagent on the binding surface and drying theprotecting reagent to form a reconstitutable dry protective layer on thebinding surface. For example, the protecting reagent is dispensed anddried prior to dispensing the liquid reagent comprising a labeleddetection reagent.

In certain specific embodiments, the binding surface is on a bottomsurface of the well and the liquid reagent is dispensed and dried on anon-overlapping bottom surface of the well or on a wall of the well.Optionally, the wall comprises a liquid storage shelf and the liquidreagent is (i) dispensed and dried on the shelf or (ii) dispensed on thewall at a location above the shelf such that liquid reagent that runsdown the wall collects and is subsequently dried on the shelf.

The methods for preparing plates may further comprise immobilizing oneor more additional capture reagents so as to form an array of bindingdomains on the binding surface that differ in their specificity oraffinity for binding partners. Similarly, the liquid reagent maycomprise one or more additional labeled detection reagents that differin their specificity or affinity for binding partners. Furthermore, themethod may include dispensing and drying an additional liquid reagentcomprising an assay control analyte with binding affinity for thecapture or labeled detection reagent, the additional liquid reagentbeing dispensed and dried such that it does not contact the capture orlabeled detection reagents.

In certain alternate embodiments of the methods described above,dispensing and drying a liquid reagent comprising a labeled detectionreagent are omitted and a reconstitutable dry labeled detection reagentis added in free-standing form, for example, as a free-standing pill.Preferably, prior to adding the detection reagent, a protecting reagentis dispensed and dried on the binding surface to form a reconstitutableprotective layer. The method may also include immobilizing one or moreadditional capture reagents so as to form an array of binding domains onsaid binding surface that differ in their specificity or affinity forbinding partners. Similarly, the reconstitutable dry reagent maycomprise one or more additional labeled detection reagents that differin their specificity or affinity for binding partners. Furthermore, themethod may include adding to the well an additional free standing dryreagent comprising an assay control analyte that has binding affinityfor the capture and/or detection reagents.

A multi-well plate is provided comprising a plate body with a pluralityof wells defined therein including: a) a plurality of first reagentwells holding a reconstitutable first dry reagent and b) a plurality ofsecond reagent wells holding a second dry reagent, wherein, the firstand second reagents are matched reagents for conducting an assay. Amethod is provided for carrying out assays in these plates comprising:a) adding a sample to one of the first reagent wells, b) reconstitutingreconstitutable dry labeled detection reagents in the first reagent wellto faint a reaction mixture, c) transferring an aliquot of the reactionmixture to one or more of the second reagent wells, and d) incubatingthe reaction mixture in the second reagent well(s) so as to carry outsaid assay on said sample. In one embodiment, the multi-well assay platecan be divided into a plurality of sets of wells consisting of one firstreagent well and one or more second reagent wells and the method furthercomprises repeating the process of (a)-(d) for each set of wells.

In one specific embodiment of the multi-well plate having first andsecond reagent wells, the first reagent wells are arranged in a regulartwo dimensional pattern and said first reagent wells have well floorsand well walls, the well walls having inner wall surfaces and outer wallsurfaces. Furthermore, the second reagent wells have well floors andwell walls, the well walls being defined by outer wall surfaces of thedetection wells and by rib elements connecting the outer wall surfacesof adjacent detection wells. Optionally, the first reagent wells havewell opening perimeters that are round and/or the first reagent wellsare arranged in an 8×12 square array.

A multi-well assay plate is provided comprising a plate body with aplurality of wells defined therein including: a) a plurality ofdetection wells, each detection well comprising a binding surface havinga capture reagent immobilized thereon and b) a plurality of reagentreconstitution wells, each reagent reconstitution well comprising areconstitutable labeled detection reagent, wherein, at least onedetection well and one reagent reconstitution well comprise matchedcapture and detection reagents for measuring an analyte of interest.Optionally, the binding surface may be selected to be suitable for useas an electrode in an electrochemical or electrochemiluminescence assay.In one embodiment, the detection and reagent reconstitution wells aregrouped into a plurality of assay sets consisting of one reagentreconstitution well and one or more detection wells, the reagentreconstitution well and detection wells within a set comprising matchedcapture and detection reagents for measuring an analyte of interest.These sets may consist of one reagent reconstitution well and onedetection well.

One specific embodiment of the multi-well plate with a detection wellsand reagent reconstitution wells includes:

-   -   a) a plurality of detection wells, wherein said detection wells,        -   i) have well floors and well walls, said well walls having            inner wall surfaces and outer wall surfaces,        -   ii) are arranged in a regular two dimensional pattern, and        -   iii) comprise, on an inner surfaces of each of said            detection wells, a binding surface having a capture reagent            immobilized thereon array;    -   b) a plurality of reagent reconstitution wells, wherein said        reagent reconstitution wells        -   i) have a well floors and well walls, said well walls being            defined by outer wall surfaces of said detection wells and            by rib elements connecting the outer wall surfaces of            adjacent detection wells, and        -   ii) comprise, in each reagent reconstitution well, a            reconstitutable dry labeled detection reagent.

Optionally, the detection wells have well opening perimeters that haveno reentrant angles or curves (e.g., round perimeters) and the reagentreconstitution wells have well opening perimeters with reentrant anglesor curves.

The detection or reagent reconstitution wells of the multi-well plateswith detection wells and reagent reconstitution wells may furthercomprise a reconstitutable dry assay control analyte. The detectionwells may also comprise one or more additional immobilized capturereagents. In this embodiment, the capture reagents are patterned tofoul′ a patterned array of binding domains on the binding surface thatdiffer in specificity or affinity for binding partners. Furthermore, thereconstitutable dry reagent may further comprise one or more additionallabeled detection reagents, the detection reagent and additionaldetection reagents differing in specificity or affinity for bindingpartners.

A method is provided for carrying out assays in multi-well plates withdetection wells and reagent reconstitution wells. One embodimentcomprises a) adding a sample to one of the reagent reconstitution wells,b) reconstituting reconstitutable dry labeled detection reagents in thereconstitution well to form a reaction mixture(s), c) transferring analiquot of the reaction mixture to one or more detection wells, c)incubating the reaction mixture in the detection well(s) underconditions that promote binding of the capture and detection reagents totheir corresponding binding partners, and d) measuring the formation ofcomplexes comprising the immobilized capture reagents and the labeledbinding reagent. Optionally, the multi-well assay plate can be dividedinto a plurality of sets of wells consisting of one first reagent welland one or more second reagent wells and the method further comprisesrepeating the process of (a)-(d) for each of said set of wells.

A multi-well assay plate is provided comprising a plate body with aplurality of wells defined therein having well floors and well wallsthat extend from said floors to a height h_(w) above said floors, saidwalls being shaped so as to provide shelf elements at a height h_(s),wherein 0<h_(s)<h_(w). The wells may be arranged in standard multi-wellplate formats including 4×6, 8×12, 16×24, and 32×48 arrays of wellsarranged in square lattices. In certain embodiments, h_(s) is greaterthan or equal to 0.02 h_(w), 0.05 h_(w) or 0.1 h_(w) but less than orequal to 0.1 h_(w), 0.25 h_(w) or 0.5 h_(w). In other embodiments, h_(s)is greater or equal to about 0.1 mm, 0.2 mm 0.5 mm, or 1 mm but lessthan or equal to about 1 mm, 2 mm, or 5 mm. The shelf elements may beused to hold dry reagents. Thus, another embodiment is a plate withreconstitutable dry reagents on the shelves. A method is provided forpreparing plates for use in an assay that comprise dispensing a liquidreagent in a well of a multi-well plate that has a shelf element anddrying the reagent to form a reconstitutable dry reagent, wherein thereagent is dispensed and dried on the shelf or dispensed on the wallabove the shelf and dried such that liquid reagent that runs down thewell wall collects on and is dried on the shelf.

In certain embodiments of the plate with wells with shelf elements, theplate body is a one-piece injection-molded part. In other embodiments,the plate body comprises a plate top having a plurality of through-holesthat define the walls of the well and a plate bottom that is sealedagainst said plate top and defines the well floors. Optionally, theplate bottom provides conductive electrode surfaces that are exposed tothe interior volume of the wells and may be used as electrodes inelectrochemical assays or electrochemiluminescence assays.

A multi-well plate is provided comprising

-   -   a) a plate body with a plurality of wells defined therein        including:        -   i) a plurality of assay wells comprising a dry assay            reagent; and        -   ii) a plurality of desiccant wells comprising a desiccant,            and    -   b) a plate seal sealed against said plate body thereby isolating        said plurality of wells from the external environment.

The plate is optionally, arranged so that the wells are in a standardwell arrangement (e.g., 4×6, 8×12, 16×24 or 32×48 arrays of wellsarranged in a square lattice). Suitable configurations of assay wellsinclude, but are not limited to, wells with dry reagents (e.g., captureand/or detection reagents) as described in the embodiments describedabove. Advantageously, the desiccant wells may be connected by dryingconduits to the assay wells, the conduits permitting diffusion of watervapor from the assay wells to the desiccant wells but intersecting thewells at a height in the assay well above the location of the dry assayreagent. In one embodiment, such conduits may be provided by sealing theplate seal against recessed channels in the top surface of the platebody that connect assay wells to desiccant wells. In certainembodiments, the wells of said plate are divided into a plurality ofassay panels comprising at least one assay well and at least onedessicant well. In these embodiments, the wells in an assay panel areinterconnected via drying conduits but are not connected to wells inother assay panels. In one specific embodiment, an assay panel comprisesone assay well and one desiccant well.

In one embodiment of a multi-well plate with assay wells and desiccantwells, the assay well comprises a binding surface having a capturereagent immobilized thereon and a reconstitutable dry labeled detectionreagent. The assay well may further comprise one or more additionalimmobilized capture reagents, the capture reagent and additional capturereagents forming a patterned array of binding domains on said bindingsurface, the binding domains differing in specificity or affinity forbinding partners. In addition, the reconstitutable dry reagent mayfurther comprise one or more additional labeled detection reagents, thedetection reagent and additional detection reagents differing inspecificity or affinity for binding partners. Optionally, the bindingsurface is suitable for use as an electrode in anelectrochemiluminescence assay.

In certain embodiments of a multi-well plate with assay wells anddesiccant wells, the plate body is a one-piece injection-molded part.Alternatively, the plate body may comprise a plate top having aplurality of through holes that define well walls and plate bottom thatis sealed against said plate top and defines well floors. Said throughholes and plate bottom may define all the wells or on only a portion ofthe wells, e.g., only said assay wells or only said desiccant wells. Theplate bottom may, optionally, provide conductive electrode surfaces thatare exposed to the interior volume of the wells.

A multi-well plate is also provided comprising

-   -   a) a plate body with a plurality of wells defined therein        containing a dry assay reagent, said plate body comprising a        plate top having a plurality of through-holes that define well        walls and a plate bottom that is sealed against said plate top        and defines well floors,    -   b) a plate seal sealed against said plate body, thereby        isolating said plurality of wells from the external environment,        and    -   c) a dessicant material.

The plate is optionally, arranged so that the wells are in a standardwell arrangement (e.g., 4×6, 8×12, 16×24, or 32×48 arrays of wellsarranged in a square). The plate bottom may, optionally, provideconductive electrode surfaces that are exposed to the interior volume ofthe wells. Suitable configurations of assay wells include, but are notlimited to, wells with dry reagents (e.g., capture and/or detectionreagents) as described in the embodiments above. In certain embodiments,the desiccant is comprised in the plate seal, a gasket layer between theplate seal and the plate top, the plate top, a gasket layer between theplate top and plate bottom and/or the plate bottom. For example, thedesiccant may be impregnated in these components or in a coating onthese components, etc. Alternatively, the plate body may define one ormore additional wells that hold the desiccant.

In one embodiment, the assay well comprises a binding surface having acapture reagent immobilized thereon and a reconstitutable dry labeleddetection reagent. Optionally, the binding surface is suitable for useas an electrode in electrochemical or electrochemiluminescence assays.The assay well may further comprise one or more additional immobilizedcapture reagents, the capture reagent and additional capture reagentsforming a patterned array of binding domains on said binding surfacethat differ in specificity or affinity for binding partners. Inaddition, the reconstitutable dry reagent may further comprise one ormore additional labeled detection reagents, the detection reagent andadditional detection reagents differing in specificity or affinity forbinding partners.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1a-1e show non-scale schematic views of several embodiments ofmulti-well plate wells that include dry reagents.

FIGS. 2a-2j show non-scale schematic top and cross-sectional views ofseveral embodiments of wells having walls with shelf elements includingledges (FIGS. 2a-2f ), bridges (FIGS. 2g-2h ) and tables (FIGS. 2i-2j )that may be used to support dry reagents.

FIGS. 3a-3c show schematic illustrations of multi-well plates havingdetection wells and reagent reconstitution wells.

FIGS. 4a-4b show top and cross-sectional schematic views of oneembodiment of a plate having detection wells and reagent reconstitutionwells, the reagent reconstitution wells being located in interstitialspaces between the detection wells.

FIGS. 5a-5f show schematic views of multi-well plates 500 (FIGS. 5a-5b), 520 (FIGS. 5c-5d ) and 540 (FIGS. 5e-5f ) having assay wells anddesiccant wells.

FIG. 6 is a schematic exploded view of one embodiment of a multi-wellassay plate.

FIGS. 7a-7c show three schematic views of a multi-well plate that isconfigured to carry out array-based multiplexed electrochemiluminescenceassays.

FIG. 8 shows one embodiment of a square well plate with dry reagentledges and seven spots per well.

FIG. 9 shows one embodiment of a square well plate with dry reagentledges, seven spots per well, and drying conduits between pairs ofadjacent wells.

FIG. 10 shows the effect of incorporating desiccant wells into assayplates on the stability of dry reagents stored in the plates.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

We describe assay modules (for example, assay plates, cartridges,multi-well assay plates, reaction vessels, etc.) having assay reagentspre-loaded in the wells, chambers, or assay regions of the assay module.In certain embodiments, these assay reagents are stored in a dry state.Furthermore, the assay modules may comprise desiccant materials formaintaining the assay reagents in a dry state. The assay modulespreloaded with the assay reagents can greatly improve the speed andreduce the complexity of assay measurements while maintaining excellentstability during storage. We also describe methods for making such assaymodules and methods for using the assay modules in assays.

The dried assay reagents may be any assay reagent that can be dried andthen reconstituted prior to use in an assay. These include, but are notlimited to, binding reagents useful in binding assays, enzymes, enzymesubstrates, indicator dyes and other reactive compounds that may be usedto detect an analyte of interest. The assay reagents may also includesubstances that are not directly involved in the mechanism of detectionbut play an auxiliary role in an assay including, but not limited to,blocking agents, stabilizing agents, detergents, salts, pH buffers,preservatives, etc. Reagents may be present in free form or supported onsolid phases including the surfaces of compartments (e.g., chambers,channels, flow cells, wells, etc.) in the assay modules or the surfacesof colloids, beads, or other particulate supports. In certainembodiments, a dry reagent (e.g., a reconstitutable dry reagent) isincluded that comprises ammonium phosphate as a buffering component,comprises other ammonium salts, and/or comprises less than about 1%(w/w) or less than about 0.1% (w/w) of sodium or potassium ions.

Many of the embodiments will be described in the context of multi-wellplates holding dry capture and detection reagents for binding assayswhere the capture and detection reagents are stored on the plate in amanner that prevents them from contacting each other. But it will beclear to the skilled artisan that such embodiments can be more generallyapplied to the storage of any number of different dry assay reagents(whether they are binding reagents, immobilized or not, labeled or not,etc.) in a manner that prevents them from contacting each other prior touse. Likewise, while many of the drawings use a “Y” symbol to representreagents or binding reagents, the use of this symbol should not beinterpreted as limiting these reagents to antibodies unless specificallystated. It will also be clear that the embodiments can be more generallyapplied to assay reagents stored in other types of assay modules incompartments other than wells (e.g., chambers, channels, flow cells,etc.).

The descriptor “reconstitutable dry” may be used to refer to dryreagents as in reconstitutable dry reagents with labeled detectionreagents or dry reconstitutable protective layers, etc. This terminologyis used to refer to dry reagents that are reconstituted by the additionof a sample or solvent to form a solution or suspension. Preferably,they are water-soluble or otherwise reconstitutable by addition of anaqueous sample. By comparison, an “immobilized” reagent, as the term isused herein, refers to the reagent that will normally remain on asurface after addition of a sample during the conduct of an assay,although there may be specific conditions that can be used to activelydissociate it from the surface.

Reconstitutable dry reagents may be prepared in situ in a compartment ofan assay module (e.g., in the well of a multi-well assay plate). By wayof example, a volume of a liquid reagent may be dispensed into the wellor other compartment and dried (e.g., by air drying, vacuum drying,freeze drying, etc.) to form the reconstitutable dry reagent. By addinga small volume that remains confined on a discrete surface of thecompartment (e.g., a discrete location on the bottom or wall of a well),the resulting dry reagent may remain fixedly confined to that location.Alternatively, a volume may be added that is sufficient to spread acrossthe bottom surface or to fill the compartment/well so as to form a dryreagent layer over the contacted surfaces. Reconstitutable dry reagentsmay be prepared outside the assay module and added to a compartment ofthe module (e.g., a well of a multi-well plate) in dry form (e.g., as adry powder or as a free-standing dry pill). Pill refers herein to acontiguous dry object such as a pressed dry tablet or a lyophilized drybead (as in U.S. Pat. No. 5,413,732).

Some embodiments include or employ dry binding reagents that are usefulin carrying out binding assays. Binding reagents that can be used in theassay modules and methods include, but are not limited to, antibodies,receptors, ligands, haptens, antigens, epitopes, mimitopes, aptamers,hybridization partners, and intercalaters. Suitable binding reagentcompositions include, but are not limited to, proteins, nucleic acids,drugs, steroids, hormones, lipids, polysaccharides, and combinationsthereof. Nucleic acids and proteins (in particular, antibodies) haveproven especially useful in binding assays. The skilled artisan will beable to identify appropriate binding reagents for a specificapplication. As used herein, the term “antibody” includes intactantibody molecules (including hybrid antibodies assembled by in vitrore-association of antibody subunits), antibody fragments and recombinantprotein constructs comprising an antigen binding domain of an antibody(as described, e.g., in Porter & Weir, J. Cell. Physiol., 67 (Suppl.1):51-64, 1966 and Hochman et al. Biochemistry 12:1130-1135, 1973). Theterm also includes intact antibody molecules, antibody fragments andantibody constructs that have been chemically modified, e.g., by theintroduction of a label. As used herein, the term nucleic acid will begenerally applied to include not only DNA and RNA but also analogs (suchas peptide nucleic acids or phosphorothioate linked nucleic acids) thatcan participate in specific Watson-Crick or Hoogstein hybridizationreactions with DNA or RNA sequences and also includes nucleic acids andanalogs that have been chemically modified, e.g., by the introduction ofa label.

The term “capture reagent” is used herein to refer to binding reagentsthat are immobilized on surface to form a binding surface for use in asolid phase binding assay. The assay modules and methods may also employor include another binding reagent, “the detection reagent” whoseparticipation in binding reactions on the binding surface can bemeasured. The detection reagents may be measured by measuring anintrinsic characteristic of the reagent such as color, luminescence,radioactivity, magnetic field, charge, refractive index, mass, chemicalactivity, etc. Alternatively, the detection reagent may be labeled witha detectable label and measured by measuring a characteristic of thelabel. Suitable labels include, but are not limited to, labels selectedfrom the group consisting of electrochemiluminescence labels,luminescent labels, fluorescent labels, phosphorescent labels,radioactive labels, enzyme labels, electroactive labels, magnetic labelsand light scattering labels.

Assays that may be carried out include “sandwich assays” that employ animmobilized capture reagent and a detection reagent that can bindsimultaneously to an analyte of interest so as to have the effect ofsequestering the detection reagent on the binding surface. Thus, thepresence of the analyte can be measured by measuring the accumulation ofthe detection reagent on the surface. Assays may also include“competitive assays” that i) employ an immobilized capture reagent thatcompetes with an analyte for binding to a detection reagent or ii) adetection reagent that competes with an analyte for binding to animmobilized capture reagent. In the case of the competitive assay, thepresence of analyte leads to a measurable decrease in the amount ofdetection reagent on the binding surface.

Capture or detection reagents may directly bind to (or compete with) ananalyte of interest or may interact indirectly through one or morebridging ligands. Accordingly, the dry assay reagents may include suchbridging ligands. By way of example, streptavidin or avidin may be usedas capture or detection reagents by employing biotin-labeled bridgingreagents that bind or compete with the analyte of interest. Similarly,anti-hapten antibodies may be used as capture or detection reagents byemploying hapten labeled binding reagents that bind or compete with theanalyte of interest. In another example, anti-species antibodies or Fcreceptors (e.g., Protein A, G or L) are used as capture or detectionreagents through their ability to bind to analyte specific antibodies.Such techniques are well established in the art of binding assays andone of average skill in the art will be able to readily identifysuitable bridging ligands for a specific application.

Certain embodiments of the assay modules/plates include a capturereagent immobilized on a surface of the module/plate so as to form abinding surface. Immobilization may be carried out using wellestablished immobilization techniques in the art of solid phase bindingassays such as the techniques that have been established for carryingout ELISA assays or array-based binding assays. In one example, bindingreagents may be non-specifically adsorbed to a surface of a well of amulti-well plate. The surface may be untreated or may have undergonetreatment (e.g., treatment with a plasma or a charged polymer) toenhance the adsorbance properties of the surface. In another example,the surface may have active chemical functionality that allows forcovalent coupling of binding reagents. After immobilizing the reagent,the surface may, optionally, be contacted with a reagent comprising ablocking agent to block uncoated sites on the surface. For conductingmultiplexed measurements, binding surfaces with arrays of differentcapture reagents may be used. A variety of techniques for forming arraysof capture reagents are now well established in the art of array basedassays.

The binding surfaces are, optionally, coated with a reconstitutable dryprotective layer. The protective layer may be used to stabilize abinding surface, to prevent a binding surface from contacting detectionreagents during manufacture or storage, or simply as a location to storeassay reagents such as bridging reagents, blocking reagents, pH buffers,salts, detergents, electrochemiluminescence coreactants, etc.Stabilizers that may be found in the protective layer include, but arenot limited to, sugars (sucrose, trehalose, mannitol, sorbitol, etc.),polysaccharides and sugar polymers (dextran, FICOLL, etc.), polymers(polyethylene glycol, polyvinylpyrrolidone, etc.), zwitterionicosmolytes (glycine, betaine, etc.) and other stabilizing osmolytes(trimethylamine-N-oxide, etc.). Blocking agents are materials thatprevent non-specific binding of assay components, especially detectionreagents, to binding surfaces and include proteins (such as serumalbumins, gamma globulins, immunoglobulins, dry milk or purified casein,gelatin, etc.), polymers (such as polyethylene oxide and polypropyleneoxide) and detergents (e.g., classes of non-ionic detergents orsurfactants are known by the trade names of BRIJ, TRITON, TWEEN, THESIT,LUBROL, GENAPOL, PLURONIC, TETRONIC, and SPAN). In certain embodiments,a protective layer is included that comprises ammonium phosphate as abuffering component, comprises other ammonium salts, and/or comprisesless than 1% or 0.1% (w/w) sodium or potassium ions.

One embodiment is a multi-well plate comprising at least one well having(1) a first dry assay reagent and (2) a second dry assay reagent whereinone or both of said first and second dry reagents is a reconstitutabledry reagent and wherein said first and second dry reagents do notcontact each other. The well may further include one or more additionaldry reagents. These may include one or more additional reconstitutabledry reagents that do not contact the first and/or second dry reagents.The embodiment also includes methods for conducting assays in theseplates for an analyte of interest comprising adding liquid samples toone or more wells of a plate, reconstituting reconstitutable dryreagents in the wells and measuring an analyte-dependent assay signal soas to measure analyte in the sample. The skilled artisan will be able toreadily select reagents and detection methodology for measuring a widevariety of analytes based on knowledge in the assay art. Detectablesignals that may be measured include, but are limited to, opticalabsorbance, photoluminescence (e.g., fluorescence), chemiluminescence,electrical current or potential, catalytic activity, chemical activity,light scattering, agglutination, radioactivity,electrochemiluminescence, magnetism, changes in refractive index, andother signals that have been used in assay measurements.

Another embodiment is a multi-well plate comprising at least one wellhaving (1) a binding surface having a first binding reagent immobilizedthereon and (2) at least one additional dry reagent, wherein at leastone additional dry reagent is a reconstitutable dry reagent that doesnot contact the binding surface. The multi-well plate may have anelectrode surface with a binding surface incorporated in at least onewell of the multi-well plate.

FIGS. 1a-1e show non-scale schematic views of several embodiments ofwell 100 of a multi-well plate. The well is defined by well floor 120and well walls 110. Floor 120 and walls 110 may be formed of a singlecontiguous material or may be separate components (e.g., a plate top andplate bottom) that are mated together. Well 100 also contains a firstdry reagent 130 located on floor 120 that, as shown, may be one or morecapture reagents that are immobilized on floor 120 to form a bindingsurface. First dry reagent 130 may include a plurality of immobilizedcapture reagents (e.g., reagents 130 a, 130 b, and 130 c) that arepatterned into a plurality of discrete binding domains (e.g., an array).Advantageously, the binding reagents/domains may have different affinityor specificity for binding partners; such binding domains may be used tocarry out multiplexed array-based measurements. A reconstitutableprotective layer 140 covers dry reagent 130. Protective layer 140 may beomitted, e.g., when it is not required to physically separate reagents130 and 150. Well 100 also comprises a second dry reagent 150 that is areconstitutable dry reagent. Second dry reagent 150 may comprise adetection reagent such as labeled detection reagent 160. Optionally,second dry reagent 150 comprises a plurality of detection reagents thatdiffer in affinity or specificity for binding partners. Well 100 mayalso include an, optional, additional reconstitutable dry reagent 170that comprises an assay control analyte 180 (as shown in FIGS. 1c-1e ).Also shown is plate seal 190. Plate seal 190, which may be omitted, issealed against the top surface of walls 110 to protect the dry reagentsfrom the environment.

FIG. 1a shows an embodiment in which first dry reagent 130 is coatedwith reconstitutable protective layer 140. Second dry reagent 150 islayered onto of protective layer 140 which prevents second dry reagent150 from contacting first dry reagent layer 130. In one example of thisembodiment, second dry reagent 150 is deposited by dispensing it inliquid form on protective layer 140; protective layer 140 is chosen tohave enough thickness or mass such that it can adsorb this liquidwithout allowing it to contact dry reagent 130. The liquid is then driedto form second dry reagent 150. In an alternate example, protectivelayer 140 is introduced in liquid form and frozen in the well to form afirst frozen layer. Reagent 150 is then introduced in liquid form andfrozen as a second frozen layer over the first frozen layer.Lyophilization of the two frozen layers provides the layered dry reagentstructure.

FIG. 1b shows an embodiment where reagents 130 and 150 are both fixedlylocated on non-overlapping regions of floor 120. Additional dryreagents, such as assay control reagents (not shown), could be locatedon other non-overlapping regions of floor 120. The localization ofreagents on selected regions of floor 120 may be carried out usingstandard techniques in patterned reagent deposition or dispensing.Optionally, floor 120 has relatively hydrophilic domains surrounded byrelatively hydrophobic areas such that appropriate volumes of reagentsdispensed on the hydrophilic domains will spread to defined boundariesdetermined by the hydrophobic areas. In this and other embodiments wherereconstitutable dry reagents are located on a surface, one may pre-treatthe surface with blocking agents to prevent adsorption of the reagentsto the surfaces and/or include blocking agents in the reagentcomposition.

FIG. 1c shows an embodiment where second dry reagent 150 is fixedlylocated, as one or more dry reagent pills, on walls 110. The pills maybe formed, e.g., by dispensing one or more droplets of the reagent (inliquid form) on walls 110 and drying them to form the dry reagent pills.FIG. 1c also shows optional additional dry reagent 170 with controlanalyte 180 fixedly located on another non-overlapping region of walls110. FIG. 1d shows an embodiment that is like that shown in FIG. 1cexcept that reagents 150 and 170 are located on shelves 115 on walls110. Dry reagents 150 and 170 may be formed from liquid reagents bydispensing and drying them on shelves 115 or dispensing them aboveshelves 115 so that they run down walls 110 onto shelves 115 where theyare dried. Alternatively, free-standing dry reagent pills may be placedon shelves 115.

Finally, FIG. 1e shows an embodiment where reagent 150 and optionalreagent 170 are free standing dry reagent pills. Also included areembodiments of well 100 in which there is some combination ofreconstitutable dry reagents on the well floor, well walls, wellshelves, and/or in free-standing form. In alternate embodiments, somecombination of fixedly located and free standing reconstitutable dryreagents is employed.

As shown in the embodiments in FIG. 1, the multi-well plates includethose having wells with multiple, physically-distinct, dry reagents.Similarly, for carrying out different assays in different wells, theremay be different dry reagents in different wells. It may be desirable,for example for QC purposes, to be sure that the correct dry reagentsare present in the wells of a plate. Accordingly, the dry reagents mayinclude indicators (such as dyes or fluorophores) that can be used inoptical inspection of the plates. By using different distinguishableindicators in different dry reagents, it is possible to opticallyinspect a plate to ensure that the correct reagents are in theappropriate locations in the appropriate wells of a plate.

FIG. 2 shows non-scale schematic views of several embodiments of wellsthat have shelf elements on which liquid reagents can be held and driedand/or on which free-standing dry reagents may be supported above thewell bottom. The shelf elements may include ledges, bridges or tables asdescribed below. FIG. 2a is a cross-section of a well 200 showing wellbottom 200 and well wall 210, the well wall having ledges such as ledges230 and 235 that can support dry reagents. Ledge 230 has an angle thatis substantially 90° or less than 90° relative to the wall directlyabove the ledge such that an appropriate volume of reagent can bedispensed on ledge 230 and accumulate on ledge 230 without overflowingonto well bottom 200. The ledges may also have additional features tohelp contain reagents such as lip 240 on ledge 235.

Shelf elements such as ledge 235 may be located at any height (h_(s))above well bottom 240 (h_(b)=0) and below the height of the well(h_(w)). In some embodiments, h_(s) is greater than or equal to 0.02h_(w), 0.05 h_(w) or 0.1 h_(w) but less than or equal to 0.1 h_(w), 0.25h_(w) or 0.5 h_(w). In other embodiments, h_(s), is greater or equal toabout 0.1 mm, 0.2 mm, 0.5 mm, or 1 mm but less than or equal to about 1mm, 2 mm, or 5 mm. Through proper selection of shelf height and volumesof sample/reagent added during the course of an assay, it may bepossible to control the order or timing of assay reactions. In oneexample, the shelf height and sample volume are chosen such thataddition of sample to the well provides a height of liquid that contactsreagents on the bottom of the well and also reconstitutes reagents onone or more shelves. Alternatively, shelf height may be chosen so thataddition of defined volume of a first liquid contacts dry reagents onthe bottom of the well (reconstituting reconstitutable reagents on thebottom and/or allowing reactions to proceed involving reagents stored onthe bottom) but does not reach the height of one or more shelves.Reactions involving reagents on the shelves can be commenced at a latertime by adding sufficient volume of a second liquid so that the liquidlevel reaches the height of the shelves so as to reconstitute dryreagent on the shelves. In conducting an assay, the sample to bemeasured may be the first liquid, second liquid or both.

FIGS. 2b-2f show top views of several embodiments of well 200 and showthat the well openings may have a variety of shapes including, but notlimited to, square (FIGS. 2b -2 d) and round (FIGS. 2e-2f ).Furthermore, the shelf elements may extend around the perimeter of thewell as in FIGS. 2b and 2e or there may be one or more isolated shelfelements that only extend partially around the well as in FIGS. 2c-2dand 2f A well may also include a plurality of shelf elements atdifferent heights within a well. FIGS. 2g-2h show cross-section and topviews, respectively, of a well 290 in which a shelf element is providedby bridge 250 that extends across the well. FIGS. 2i-2j showcross-section and top views, respectively of a well 295 in which a shelfelement I provided by a table 260 that extends vertically from an areaof well bottom 220.

A multi-well plate is provided comprising a plate body with a pluralityof wells defined therein including: a) a plurality of first reagentwells holding a reconstitutable first dry reagent and b) a plurality ofsecond reagent wells holding a second dry reagent (which may be areconstitutable dry reagent or an immobilized reagent), wherein, thefirst and second reagents are matched reagents for conducting an assay(i.e., they are both used in conducting an assay of interest). Thereagents may be located in a variety of locations with the wells such aswell bottom, well walls, on shelf elements, as free-standing pills orpowders, etc. A method is provided for carrying out assays in theseplates comprising: a) adding a sample to one of the first reagent wells,b) reconstituting reconstitutable dry labeled detection reagents in thefirst reagent well to form a reaction mixture, c) transferring analiquot of the reaction mixture to one or more of the second reagentwells, and d) incubating the reaction mixture in the second reagentwell(s) so as to carry out said assay on said sample. In one embodiment,the multi-well assay plate can be divided into a plurality of sets ofwells consisting of one first reagent well and one or more secondreagent wells and the method further comprises repeating the process of(a)-(d) for each set of wells.

FIG. 3a is a (not to scale) schematic illustration of one embodimentshowing cross-sectional views of two wells of a multi-well plate 300.Well 302 is a reagent reconstitution well comprising one or morereconstitutable dry reagents which may include a labeled detectionreagent (such as dry reagent 350 comprising labeled detection reagent360) or a an assay control analyte (such as dry reagent 370 comprisingassay control analyte 380). These dry reagents may include additionalreagent components such as blocking agents, stabilizers, preservatives,salts, pH buffers, detergents, bridging reagents, ECL coreactants andthe like. The reagents may be located on well bottoms, specificlocations on well bottoms, on well walls, shelf elements or may befree-standing (as per the discussion of FIGS. 1 and 2). Well 301 is adetection well comprising one or more dry reagents which may includereconstitutable dry reagents or an immobilized dry reagent. As shown,well 301 comprises immobilized capture reagents 330 that are patternedinto three binding domains 330 a, 330 b, and 330 c to form a bindingsurface. Well 301 also comprises a reconstitutable protective layer 340which may be omitted. In one embodiment of an assay, sample is added tothe reagent reconstitution well where reconstitutable dry reagents arereconstituted. The sample is then transferred to the detection wellwhere the assay measurement is carried out. Alternatively, areconstitution buffer may be used to reconstitute reagents in thereagent reconstitution well; the reconstitution buffer is then combinedwith sample in the detection well. FIG. 3a also shows plate seal 390which seals against the openings of wells 301 and 302 to protect thecontents of the wells from the environment.

The detection and reagent reconstitution wells in a multi-well plate maybe grouped into a plurality of assay sets consisting of one reagentreconstitution well and one or more detection wells, the reagentreconstitution well and detection wells within a set comprising matchedcapture and detection reagents for measuring an analyte of interest.FIG. 3b shows an arrangement where a set has one reagent reconstitutionwells 302 and three detection wells 301. During an assay, a sample addedto well 302 may then be distributed among the three associated detectionwells 301 so as to conduct multiple replicates or, where the detectionwells hold different reagents, multiple different assays. FIG. 3c showsan arrangement where a set has one reagent reconstitution well 302 andone detection well 301.

Reagent reconstitution wells and detection wells may be similar in sizeand shape or may have different sizes and/or shapes. In some embodiment,the wells in a standard multi-well plate are divided between the twotypes of wells. FIG. 4 shows a non-scale schematic views of analternative arrangement of wells. FIG. 4a shows a top view of multi-wellplate 400 having detection wells 440 that are arranged in a regular twodimensional pattern and that have detection wells walls 430 with innerwall surfaces and outer wall surfaces. Multi-well plate also has reagentreconstitution wells 460 in interstitial spaces between detection wells.Reagent reconstitution wells 460 have well walls that are defined by theouter well surfaces of detection well walls 430 and rib elements 450that connect the outer surfaces of well walls 430 of adjacent detectionwells (and, in the outermost of the wells, by the inner surface of plateframe wall 410). As shown, the detection wells may be shaped to have noreentrant (i.e., inward pointing) curves or angles while theinterstitial wells may have reentrant curves and/or angles. FIG. 4bshows a cross-sectional view along the dotted line in FIG. 4a and showsthe bottom surfaces of the two types of wells (which may be at differentheights in the plate body). Plate 400 may be formed from a singlecontiguous material. In an alternate embodiment, plate 400 is formedfrom a plate top 405 and a plate bottom 420 that are mated along thedotted line shown in FIG. 4b . Advantageously, the basic arrangement ofarrays of round wells with interstitial wells defined by the well wallsand rib elements is a common feature of many injection-molded 96-wellplates and plate tops and allows these components to be used to form dryreagent plates as shown in FIG. 4.

A multi-well plate is provided comprising a) a plate body with aplurality of wells defined therein including: i) a plurality of assaywells comprising a dry assay reagent; and ii) a plurality of desiccantwells comprising a desiccant, and b) a plate seal sealed against saidplate body thereby isolating said plurality of wells from the externalenvironment. In some embodiments, the assay wells comprise the necessaryreagents for conducting an assay in the assay well. Also included areembodiments in which the desiccant wells are connected by dryingconduits to the assay wells, the conduits permitting diffusion of watervapor from the assay wells to the desiccant wells but intersecting thewells at a height in the assay well above the location of the dry assayreagent. In addition to multi-well plates containing dry reagents anddesiccants, the plates themselves (i.e., without dry reagents anddesiccants), in particular, plates having conduit or channel elements(e.g., as shown in FIG. 5 described below) that are suitable forconnecting sets of desiccant and assay wells with dry reagents areprovided.

FIG. 5 shows non-scale schematic views of a multi-well plate 500 havingassay wells 501 and desiccant wells 502 (desiccant and dry reagents arenot shown). FIG. 5a is a top view showing well walls 510 and conduits508 connecting dessicant wells with one (e.g., as in row A) or moreassay wells (e.g., as in row B). FIG. 5b shows a cross-sectional viewalong the dotted line in FIG. 5a and together with FIG. 5a shows howconduits 508 may be formed by sealing plate seal 515 against channels inthe top surface of the plate body. Plate seal 515 seals against thesechannels and the tops of the wells to form sets of assay and dessicantwells that are interconnected by conduits but are isolated from theenvironment and from other sets of wells. Accordingly, one or more setsof wells may be unsealed and used in an assay and the remaining sets ofwells will be maintained in a dry environmentally protected environment.Plate 500 may be formed from a single contiguous material. In analternate embodiment, plate 500 is formed from a plate top 505 and aplate bottom 512 that are mated along the dotted line shown in FIG. 5b ,plate bottom 512 defining the floor of at least some of the wells.

The assay wells or sets of wells in plate 500 may include one or more ofany of the dry reagent-containing wells described above, for example, inthe descriptions of FIGS. 1-4 and may include both detection wells andreagent reconstitution wells. The desiccants used in the desiccant wellmay be selected from known desiccant materials including, but notlimited to, silica, activated alumina, activated clays, molecular sievesand other zeolites, hydroscopic salts (e.g., anhydrous calcium sulfate,magnesium sulfate, sodium sulfate, sodium hydroxide and lithiumchloride), hydroscopic solutions (e.g., concentrated solutions oflithium chloride) and water reactive materials such as phosphorouspentoxide. In some embodiments, the desiccant is present as a free drypowder or granular material. In other embodiments, the desiccant ispresent as a dry pill, for example a pressed tablet or a desiccantimpregnated polymeric material. In other embodiments, the desiccant iscontained in a water vapor permeable bag or container (e.g., as incommercial silica pouches). Advantageously, desiccant in pill form orpre-packaged containers may be “press fit” into desiccant wells toprevent movement in the well during shipping or use.

FIGS. 5c-5d show top and cross-sectional views of one embodiment of amulti-well plate 520 with assay and desiccant wells. Plate 520 has assaywells 521 (which may contain dry assay reagents) that are arranged in aregular two dimensional pattern and that have assay well walls 523 withinner wall surfaces and outer wall surfaces. It also has desiccant wells522 in interstitial spaces between detection wells (alternatively, wells521 are used as desiccant wells and wells 522 are used as assay wells).Desiccant wells 522 have well walls that are defined by the outer wellsurfaces of detection well walls 523 and rib elements 525 that connectthe outer surfaces of well walls 523 of adjacent assay wells (and, inthe outermost of the wells, by the inner surface of plate frame wall526). Channels 524 notched into the top of well walls 523 provide, whenmated to a plate seal, paths for water vapor to travel from assay wellsto desiccant wells. As shown, the assay wells may be shaped to have noreentrant (i.e., inward pointing) curves or angles while theinterstitial wells may have reentrant curves and/or angles. FIG. 5dshows a cross-sectional view along the dotted line in FIG. 5c and showsplate seal 527 which is mated to the top of the plate to form sets ofassay and desiccant wells that are connected via conduits 524 butisolated from other wells and from the environment. Plate 520 may beformed from a single contiguous material. In an alternate embodiment,plate 520 is formed from a plate top 528 and a plate bottom 529 that aremated along the dotted line shown in FIG. 5 d.

FIG. 5e shows a schematic view of another embodiment of a multi-wellplate with assay wells (which may contain dry reagents) and desiccantwells and shows a plate 540 with assay wells 541 and desiccant wells 543that are connected into sets of wells via channels 542 in the platebody. Multi-well plate 540 is largely analogous to the embodiment ofplate 500 pictured in FIGS. 5a-5b except that in plate 540, desiccantwells 542 are much shallower and smaller in area than the assay wellsallowing a larger portion of the plate footprint to be dedicated towells used in assay measurements. FIG. 5f shows a cross-sectional viewalong the dotted line in FIG. 5e and also shows plate seal 544 that issealed against the top of the plate to form connected sets of assay anddesiccant wells. Plate 540 may be formed from a single contiguousmaterial. In an alternate embodiment, plate 540 is formed from a platetop 545 and a plate bottom 546 that are mated along the dotted lineshown in FIG. 5f , plate bottom 546 also defining the floor of assaywells 541.

FIG. 6 is a schematic exploded view of one embodiment of a multi-wellassay plate. Multi-well assay plate 600 comprises a plate top 610 withthrough-holes 615 that define the walls of wells. Plate top 610 issealed against plate bottom 620 through gasket 625 such that platebottom 620 defines the bottom surface of the wells. Optionally, platetop 610 is sealed directly to plate bottom 620 and gasket 625 isomitted. Sealing may be accomplished through traditional sealingtechniques such as adhesives, solvent welding, heat sealing, sonicwelding and the like. In another optional embodiment, plate top 610fully defines the sides and bottom of the wells and plate bottom 620 andgasket 625 may be omitted. The contents of the wells, which may includewells configured to contain dry reagent and/or desiccant as describedabove, may be protected from the outside environment by sealing (e.g.,via traditional sealing techniques) plate seal 630 to plate top 610directly or via optional gasket 635.

The components of plate 600 may be made from a variety of differentmaterials including, but not limited to, plastics, metals, ceramics,rubbers, glasses or combinations thereof. In accordance with therequirements of the particular detection technology used with theplates, the components some or all of the components may be selected tobe transparent, colored, opaque, or highly light scattering. In oneembodiment, plate top 610 is an injection-molded plastic such asinjection-molded polystyrene, polypropylene, or cyclic olefin copolymer(COC). Optionally, one or more of the components may be made of orcomprise (for example in the form of a coating) a material that has alow water vapor transmission rate, e.g., a water vapor transmission rateless than 1 g/m² per day through a 100 um thickness. Low water vaportransmission materials include, but are not limited to, glass, metals ormetal films (e.g., aluminum films), COC, polyvinylidene chloride (PvDC),polypropylene, polychlorotrifluoroethylene (PCTFE), and liquid crystalpolymers (LCP).

Plate 600 may include desiccant wells as described above. Alternatively,or in addition, desiccant may be incorporated directly into plate top610, plate bottom 620, plate seal 630, gasket 625 and or gasket 635. Forexample, U.S. Pat. No. 6,174,952 to Hekal et al. describes desiccantcontaining polymer blends that may be molded, cast into liners, orformed into films, sheets, beads or pellets.

In some embodiments, plate bottom 620 has features to facilitate thepatterning of reagents on the bottom of wells (e.g., patternedhydrophilic features surrounded by hydrophobic areas) and/or conductivelayers that provide electrodes that are exposed to the interior volumesof the wells of plate 600 so that electrochemical or electrode inducedluminescence assays (e.g., electrochemiluminescence assays) may becarried out. Plate bottom 620 may also include electrode contacts toallow an external instrument to apply electrical potential/current tothe electrodes. Suitable approaches, configurations and compositions forsuch features, conductive layers and electrode contacts include thosedescribed in U.S. Publications 2004/0022677 and 2005/0052646 toWohlstadter et al. Suitable instrumentation and methods that can be usedto conduct ECL measurements using assay modules include those describedin U.S. Publications 2004/0022677 and 2005/0052646 of U.S. applicationSer. Nos. 10/185,274 and 10/185,363, respectively; U.S. Publication2003/0113713 of U.S. application Ser. No. 10/238,391; U.S. Publication2005/0142033 of U.S. application Ser. No. 10/980,198; and theconcurrently filed U.S. application Ser. No. 11/642,968 of Clinton etal. entitled “Assay Apparatuses, Methods and Reagents.”

FIG. 7 provides schematic illustrations of one specific embodiment thatincludes some of the inventive concepts disclosed above in the contextof a multi-well plate configured to carry out an-ay-based multiplexedelectrochemiluminescence assays. FIG. 7a shows a section of multi-wellplate 700 that has a plurality of assay wells 710 which may comprise dryreagents and a plurality of desiccant wells 720 which may comprise adesiccant. Channels 725 on the top surface of plate 700 link eachdesiccant well to an assay well. Optionally, desiccant wells 720 andchannels 725 are omitted. Assay wells 710 have ledges 712 which may beused to support a reconstitutable dry reagent (e.g., dry reagentscomprising assay controls and/or ECL labeled detection reagents). Assaywells also have working electrode surfaces 714 which are covered bypatterned dielectric layer 716 so as to expose a plurality of exposedelectrode surfaces or “spots” (shown as circles within the wells). Inaddition, counter electrodes 718 are provided to provide for a completeelectrochemical circuit. Optionally, the surface of dielectric layer 716is hydrophobic relative to electrode surface 714 so that small volumesof reagents patterned onto the spots may be kept confined to the spots.The different spots may have different capture reagents immobilizedthereon to form a binding surface with an array of binding domainsdiffering in specificity or affinity for binding partners (e.g.,analytes of interest). Alternatively, some of the spots may havereconstitutable dry reagents confined thereon which, e.g., may containassay controls and/or ECL labeled detection reagents. The assay well mayfurther comprise a reconstitutable protective layer over the bindingsurface.

FIG. 7b provides an exploded cross-sectional view along the dotted linein FIG. 7a and illustrates one approach to forming theelectrode/dielectric layers in assay wells 710. The multi-well platecomprises a plate top 730 that defines desiccant wells 720 and hasthrough-holes that define the walls of assay wells 710 and ledges 712.Plate top 730 also has channels 725 that form conduits between assaywells 710 and desiccant wells 720 when plate seal 750 is sealed againstthe top surface of plate top 730. In one non-limiting example, plate top730 is an injection-molded part molded from a plastic with low watervapor transmission. In another non-limiting example, plate seal 750 is aheat sealable film comprising a low water vapor transmission plastic ora metal (e.g., aluminum) foil.

FIG. 7b also shows plate bottom 740 which seals against plate top 730and defines the bottom of assay wells 710. Plate bottom 740 comprisessubstrate 715 which supports patterned conductive layers that providefor electrodes 714 and 718. Patterned dielectric layer 716 on theelectrodes defines the exposed electrode spots. A variety of materialsmay be used to provide for the substrate and the conductive anddielectric layers (see, e.g., U.S. Publications 2004/0022677 and2005/0052646). In one non-limiting example, the substrate is a plasticfilm (made, e.g., of a polyester such as MYLAR, polyvinylchloride, or alow water vapor transmissive material such as COC), the conductivelayers are screen printed conducting inks (e.g., screen printed carboninks) and the dielectric layer is a screen printed insulating ink. Alsoshown in FIG. 7b are electrode contacts 780 and 785 which are conductivelayers on the bottom of substrate 715 that provide connectivity (e.g.,via conductive through holes in substrate 715 to electrodes 714 and 718.The electrode contacts may also be provided by screen printed conductiveinks which during printing can be caused to fill holes in substrate 715to also provide the conductive through-holes. Advantageously, theconductive through-holes may be located directly below well walls tolimit water vapor transmission through the holes. In addition, anoptional bottom sealing layer 790 may be sealed to the bottom ofsubstrate 715. Bottom sealing layer 790 is made of a low water vaportransmissive material and covers most of the bottom surface of substrate715 except for defined openings in sealing layer 790 that are located soas to allow a plate reading instrument to contact electrode contacts 780and 785.

FIG. 7c shows a more detailed angled view of one embodiment of plate 700and shows desiccant pills 722 that are press-fit into desiccant wells720.

A variety of samples which may contain an analyte or activity ofinterest may be assayed. In one example, a sample is introduced to anassay plate or one or more wells of an assay plate havingreconstitutable dry reagents pre-loaded thereon, thus reconstitutingthese assay reagents and an assay signal is measured so as to measure(quantitatively or qualitatively) the amount of analyte in the sample.The reagents may include a luminescent, electrochemiluminescent,chemiluminescent, and/or redox-active substance. Accordingly, the assaysignal is preferably a luminescent or electrochemical signal. Assaysformats that may be carried out include homogeneous and heterogeneousmethods.

Assays that may be carried out include formats that employ solid-phasesupports so as to couple the measurement of an analyte or activity tothe separation of labeled reagents into solution-phase and solid phasesupported portions. Examples include solid-phase binding assays thatmeasure the formation of a complex of a material and its specificbinding partner (one of the pair being immobilized, or capable of beingimmobilized, on the solid phase support), the formation of sandwichcomplexes (including a capture reagent that is immobilized, or capableof being immobilized, on the solid phase support), the competition oftwo competitors for a binding partner (the binding partner or one of thecompetitors being immobilized, or capable of being immobilized, on thesolid phase support), the enzymatic or chemical cleavage of a label (orlabeled material) from a reagent that is immobilized, or capable ofbeing immobilized on a solid phase support and the enzymatic or chemicalattachment of a label (or labeled material) to a reagent that isimmobilized or capable of being immobilized on a solid-phase support.The term “capable of being immobilized” is used herein to refer tobridging reagents that may participate in reactions in solution andsubsequently be captured on a solid phase during or prior to detection.For example, the reagent may be captured using a specific bindingpartner of the reagent that is immobilized on the solid phase.Alternatively, the reagent is linked to a capture moiety and a specificbinding partner of the capture moiety is immobilized on the solid phase.Examples of useful capture moiety-binding partner pairs includebiotin-streptavidin (or avidin), antibody-hapten, receptor-ligand,nucleic acid—complementary nucleic acid, etc.

In assays carried out on solid-phase supports, the amount of analyte oractivity may be determined by measuring the amount of label on the solidphase support and/or in solution using i) a surface selective technique,ii) a solution selective technique and/or iii) after separation of thetwo phases. In electrochemiluminescence methods, the solid phase supportmay also be the working electrode used to induceelectrochemiluminescence from labels bound to the solid phase. Theelectrochemiluminescence methods may include washing to remove unboundelectrochemiluminescence labeled reagents prior to addition of an ECLcoreactant (e.g., tertiary amines such as tripropylamine orpiperazine-1,4-bis(2-ethanesulfonic acid)) and application of apotential to induce ECL from bound labels. Alternatively, because of thesurface selectivity of electrochemiluminescence measurements, the methodmay be ran without washing. Advantageously, in unwashed assays, the ECLcoreactant may be pre-added to assay wells in the form of areconstitutable dry reagent or protective layer.

Another embodiment relates to kits for use in conducting assays thatcomprise the assay modules/multi-well plates. The kit may include one ormore additional reagents in one or more containers including, but notlimited to, assay calibrators, assay controls, assay diluents, ECLcoreactants, and wash buffers.

According to one embodiment, the kit comprises one or more of the assaycomponents in one or more plate wells, preferably in dry form. In onepreferred embodiment, the kit comprises an assay plate having a bindingimmobilized on one or more working electrodes within an assay module andone or more additional assay reagents deposited in the form of a drybead, pellet or a pill directly into the well, preferably at a positionspacially separated from a working electrode, or alternatively depositedinto one or more interstitial wells. Preferably, the kits do not containany liquids in the wells.

Examples

The following examples are illustrative of some of the methods andinstrumentation falling within the scope of the present invention. Theyare, of course, not to be considered in any way limiting of theinvention. Numerous changes and modifications can be made with respectto the invention by one of ordinary skill in the art without undueexperimentation.

Materials & Methods Labeled Detection Antibodies

Labeled detection antibodies were labeled with SULFO-TAG NHS ester (MesoScale Discovery, Gaithersburg, Md.), an electrochemiluminescent labelbased on a sulfonated derivative of ruthenium-tris-bipyridine (compound1 pictured below). Labeled antibodies were purified by size exclusionchromatography on SEPHADEX G-50 (Pharmacia).

Lyophilized Detection Antibody Pills

Pills comprising one or more labeled detection antibodies were formedfrom a solution containing 1 μg/mL of each of the labeled antibodies, 2%bovine serum albumin and 20% sucrose in a phosphate buffered saline.Frozen droplets of this solution were formed by dispensing 20 μLdroplets into liquid nitrogen. The frozen droplets were transferred ontochilled (≤−78° C.) aluminum trays which were placed on the shelves of anADVANTAGE XL lyophilizer (Virtis). The shelves of the lyophilizer werepre-cooled to ≤−45° C. prior to introduction of the aluminum trays and aconductive paste was used to improve the heat transfer between theshelves and the trays containing beads. In a typical lyophilizationprotocol, the lyophilizer chamber was evacuated and the shelftemperature was slowly increased to −30° C., −20° C., −15° C. andfinally to +20° C. (ambient conditions) over the course of about 24hours. The temperature was held at each of these levels for sufficienttime to allow for equilibration while controlling the chamber pressure0.01 torr. Karl Fisher titrations of lyophilized beads typically showedwater contents of less than 4% by weight. The water content could bereduced to under 2% by extended storage in the presence of a dessicant.

Multi-Well Plates for Electrochemiluminescence Measurements

Electrochemiluminescence measurements were carried out using speciallydesigned multi-well plates having integrated screen printed carbon inkelectrodes for carrying out electrochemiluminescence measurements(MULTI-ARRAY or MULTI-SPOT plates, Meso Scale Discovery, a division ofMeso Scale Diagnostics, LLC, Gaithersburg, Md.). A patterned dielectriclayer patterned over the working electrode on the bottom of each wellexposes one or more regions or “spots” on the working electrode. In someexperiments, the electrode surfaces were treated with an oxygen plasmaprior to immobilizing antibodies on them. Different capture antibodieswere immobilized on the different spots by patterned micro-dispensing ofsolutions of the antibodies on the spots using a nanoliter dispenser(Bio-Dot, Inc.). The volumes dispensed on the spots were selected sothat they spread to the boundaries defined by the dielectric layers butremained confined on the spots, thus allowing for the immobilization ofeach antibody (via passive adsorption) on a defined region of theworking electrode; if the electrode surfaces were not plasma treated, asmall amount of TRITON X-100 detergent was added to the spottingsolutions to enhance spreading. Adsorption was allowed to proceed for atleast 2 hours after which the plates were washed with a stabilizing washbuffer (2% sucrose, 185 mM dibasic ammonium phosphate, 13 mM monobasicammonium phosphate, 0.1% TWEEN 20, and KATHON CG/ICP II preservative),dried, and stored in the presence of a desiccant. By controlling theamount of wash buffer left in the wells before drying (typically between5-20 μL), sucrose films of different thicknesses could be left over theworking electrode surfaces.

Electrochemiluminescence Measurement Instrument

Electrochemiluminescence was induced and measured in the MULTI-SPOTplates using a Sector® Imager 6000 reader or a Sector® PR 400 reader(both from Meso Scale Discovery, a division of Meso Scale Diagnostics,LLC, Gaithersburg, Md.). The Sector® Imager 6000 instrument applieselectrical potentials to the working electrodes in the plate and imagesthe resultant ECL. Image analysis algorithms are employed to distinguishand quantitated the light emitted from each spot in a well. The Sector®PR 400 instrument applies electrical potential to the working electrodesin one column of the plate at a time. An array of photodiodes is used tomeasure the ECL emitted from the wells in the column.

Example 1. Multiplexed Cytokine Detection Using Labeled DetectionAntibodies in Lyophilized Beads

High binding MULTI-SPOT plates having a 7 spot array of captureantibodies against seven different human cytokines (TNF-α, IL1-β, IL2,IL5, IL6, IL8, IL12, and GM-CSF) and lyophilized beads containinglabeled detection antibodies against the same seven cytokines wereprepared as described above. One bead was placed in each well and theplates were stored in the presence of dessicant until used. Multiplexedcytokine assays were carried out by introducing cytokine solutions (40μl per well prepared in RPMI cell culture media supplemented with 10%fetal calf serum) of pre-defined concentrations into the wells of theplate and incubating for 2 hours at room temperature on a plate shaker.MSD® READ BUFFER P (Meso Scale Discovery), a solution containing atertiary amine ECL coreactant, was added at 2× concentration to thewells (110 μl/well) and the plate was analyzed on a Sector® Imager 6000instrument. The resultant signals on each spot showed good linearity forall seven cytokines between 10 and 10,000 pg/ml. The standard deviationsof the signals were typically less than 10% of the average signals.Background signals and calculated sensitivities were similar to thoseobtained when the antibodies were added to the wells as liquidsolutions.

Example 2. Cytokine Measurements Using a Labeled Detection Antibody thatis Dried on a Protective Layer Covering a Capture Surface

This assay used a small spot MULTI-ARRAY plate with a single spot perwell. The spot treated, as described in the Materials and Methodssection with a solution containing an anti-human TNF-α capture antibodyto immobilize the antibody on the spot surface. The well was then filledwith 75 μL of 4×MSD® READ BUFFER P that was supplemented with 7% FICOLL(a highly branched hydrophilic polymer of sucrose), and the plate wascooled to freezing and lyophilized overnight to provide a protective“cake” layer over the bottom of the well. A small droplet (35 nL) of aconcentrated solution of a labeled anti-human TNF-α detection antibodywas dispensed on the surface of the cake. The plate was then vacuumdried for 5 minutes and stored in the presence of dessicant until used.Assays were carried out by adding to the wells 150 μL of solutionscontaining pre-determined concentrations of human TNF-α in RPMI cellculture media supplemented with 10% fetal calf serum and shaking for twohours. The plate was then analyzed on a Sector® Imager 6000 instrument.The calculated detection limits of 5-6 pg/mL are comparable to thoseobserved in non-washed assays using liquid detection antibody solutions.

Example 3. Cytokine Measurements Using a Labeled Detection Antibody thatis Dried on the Sides of the Wells of a Multi-Well Plate

This assay used a small spot MULTI-ARRAY plate with a single spot perwell. The spot was coated, as described in the Materials and Methodssection with an anti-human TNF-α capture antibody. Droplets (1 μL) of a24 μg/mL solution of the detection antibody in 4.8% sucrose weredispensed on the inside walls of the wells and allowed to dry. Theplates were stored in the presence of dessicant until used in an assay.The assay protocol involved adding 80 μL of a TNF-α solution to eachwell, shaking the plate for 30 minutes at room temperature, washing theplate, adding 150 μL of 1×MSD® READ BUFFER T (Meso Scale Discovery) andanalyzing the plate on a Sector® Imager 6000 instrument. Plates storedfor 18 days at room temperature or 4° C. gave detection limits that wereless than 1 pg/mL and comparable to those observed in washed assaysemploying liquid detection antibody solutions.

Example 4. Cytokine Measurements Using a Multi-Well Plate with WellsHaving a Capture Layer Coated with a Protein-Containing Protective Layerand Dried Labeled Detection Antibody on the Well Walls

This assay used a MULTI-ARRAY plate with a single spot per well. Theworking electrode in each well was pre-coated with streptavidin(streptavidin MULTI-ARRAY plate, Meso Scale Discovery). Anti-IL1-βmonoclonal antibody was immobilized on the working electrode accordingto the following protocol. The wells were washed three times with PBSand then treated with 20 μL of a 3 μg/mL solution of biotin-labeledanti-IL1-β. The immobilization was allowed to proceed over 2 hours underagitation on a plate shaker. The wells were then washed three times withPBS. A 20 μL volume of a buffered solution of BSA and sucrose was addedto the wells and then dried in the wells under vacuum to form a dry filmon the bottom of the plates.

A dry pill of SULFO-TAG labeled anti-IL1-β polyclonal antibody wasformed on the well wall according to the following protocol. A 100 nLmicrodroplet of a 482 μg/mL solution of the labeled antibody wasdispensed on each well wall using a BIO-DOT microdispensor (Bio-Dot,Inc.) with an angled tip. The droplet remained on the well wall where itwas allowed to dry for 30 minutes in a dessicator chamber. The wellswere then sealed with a plate heat seal. In some experiments, a lowconcentration of fluorescein was added to the detection antibodysolution. The fluorescein fluorescence could be used to provide aquality control check by identifying any well in which the detectionantibody ran down the well wall or splattered on the well bottom. Thefluorescein did not affect assay performance.

Assays for IL1-β were carried out by adding 125 μL of solutionscontaining known amounts of IL1-β to the wells and incubating for 37minutes while shaking the plate. The plate was then washed with PBS, MSDREAD BUFFER T was added and the plate was analyzed on a Sector® PR 400instrument. Assays using plates with dry detection antibodies performedin a comparable fashion to assays that used liquid detection antibodysolutions.

Example 5. Assays in Multi-Well Plates Using Dry Reagents: Storage ofDried Labeled Antibodies on Ledges in the Wells

This assay used a Multi-Spot plate configured as shown in FIG. 8. Theplate was similar to that shown in FIG. 7 except for the use of a 7-spotpattern and the omission of desiccant wells 720, channels 725, desiccantpills 722, and bottom sealing layer 790. The plate top wasinjection-molded polypropylene. Capture antibodies against wereimmobilized by dispensing, on the individual spots, antibodies againstbotulinum toxin A (BotA), dinitrophenyl (DNP), ricin, staphylococcalenterotoxin B (SEB), Venezuelen equine encephalitis (VEE), and Yersiniapestis (YP). Non-immune mouse IgG was immobilized on the remaining spotfor use as a negative control. Immobilization was carried out bydispensing 75 nL of solutions comprising between 100-500 μg/mL of anantibody, 750 μg/mL of BSA, and 0.03% TRITON X-100. One exception wasthe BotA capture antibody which was biotinylated and immobilized afterpre-binding it to 1200 μg/mL avidin and which was immobilized in theabsence of BSA.

The non-immune IgG should not participate in a sandwich complex andshould give a low signal for all samples. Elevation of this signaloutside of a selected range can be used as an indication that ameasurement artifact is producing elevated non-specific binding ofdetection antibodies and that there is a risk of false positive results.More generally, any binding reagent that is not paired with acorresponding detection reagent may be used. Optionally, the bindingreagent may be selected to share structural properties with the testcapture reagents, for example, in an immunoassay it may includeimmunoglobulins from one or more of the species from which the othercapture antibodies were derived. The anti-DNP spot will be used as apositive control. The well will also include a dry SULFO-TAG labeledanti-fluorescein (FL) antibody and a defined quantity of dry BSA labeledwith both DNP and FL (DNP-FL-BSA). The positive control signal should,therefore give a constant positive signal indicative of the definedquantity of DNP-FL-BSA. Reduction of this signal below a selected rangecan be used as an indication that a sample interferes with bindingreactions or signal generation and that there is a risk of falsenegative results. More generally, the positive control may be an assayfor any analyte that can be spiked into the reaction mixture.Preferably, there is a low likelihood of finding the analyte in thesamples of interest.

The capture antibody solutions were allowed to dry for 30 minutes in adesiccated environment and then dried for 30-60 minutes under vacuum.The wells were washed with a the stabilizing wash buffer containingsucrose described in the Materials and Methods section, blocked with 5%BSA for 45 minutes and washed once more with the stabilizing washbuffer. A stabilizing/blocking solution (20 μL of 305 mM ammoniumphosphate, 100 mM ammonium chloride, 0.02% TRITON X-100, 2% sucrose, 2%BSA, and 0.02% KATHON preservative, pH 7.4) was added and the solutionwas dried in the well under vacuum to form a dry reagent cake on thewell bottom.

A mixture of STAG-labeled detection antibodies (0.5 μL of a mixture ofantibodies against BotA, FL, ricin, SEB, VEE, and YP at between 40-240μg/mL in the stabilizing/blocking solution) was dispensed (using aBIO-DOT dispenser with an angled dispense tip) on the walls of the wellsjust above the dry reagent ledge and allowed to flow down onto theledge. A solution containing 80 ng/mL of the positive control analyte(DNP-FL-BSA) was dispensed on the opposite wall. The detection antibodyand control solutions were allowed to dry for 30-60 minutes undervacuum. The plates were then packaged with desiccant until used.

The protocol used to conduct assays with these plates was: add 80 μL ofsample (defined amounts of one or more of the target analytes in 0.1%TRITON X-100 in phosphate buffered saline (PBS)), incubate 1 hour withshaking, wash with PBS, add 150 μL 1×MSD READ BUFFER T (Meso ScaleDiagnostics, LLC) and analyze plate using an MSD Sector® Imager 6000instrument. VEE and YP used in this assay were inactivated byirradiation. BotA was used in the assay was inactivated with formalin.

The table below shows that the signals observed at each spot for samplescontaining no analyte (−) or for samples containing 10 ng/mL BotA, 1ng/mL ricin, 50 ng/mL SEB, 1000 ng/mL VEE, or 10,000 CFU/mL YP. Thetable shows sensitive and specific detection of the target analytes andproper performance of the positive and negative control spots.

Capture Spot Analyte BotA Ricin SEB VEE YP Neg Pos BotA 28893 178 97 182159 134 9456 Ricin 243 15502 106 222 142 129 8776 SEB 276 165 9518 230177 162 8288 VEE 1516 233 188 11821 204 237 8506 YP 243 129 107 211 4280152 8656 — 249 81 75 212 115 121 8923

The next table compares the signals observed with these plates to assayscarried out under comparable conditions except for the use of liquiddetection reagents. The table provides only the signal on the specificspot for a given analyte and provides both signal in the presence ofanalyte (10 ng/mL BotA, 1 ng/mL ricin, 50 ng/mL SEB, 1000 ng/mL VEE, or10,000 CFU/mL YP) and background signal in the absence of analyte. Thetable shows that the dry and wet assays perform comparably.

Signal on Specific Spot Dry Wet Analyte Backrd Signal Backrd Signal BotA249 12098 156 4809 Ricin 81 15502 94 12531 SEB 75 9518 68 6299 VEE 21211821 260 7497 YP 115 4280 145 3876

Example 6. Assays in Multi-Well Plates with Assay Wells and DesiccantWells

This assay used a MULTI-SPOT plate configured as shown in FIG. 9. Theplate was similar to that shown in FIG. 8 except for the inclusion ofconduits 910 connecting pairs of adjacent wells. The conduits wereprovided by shallow notches that were cut into the walls separatingadjacent wells. In this example, one well of each pair of wells was usedto carry out a multiplexed immunoassay and the other was used to holddesiccant for maintaining the assay well in a dry state during storage.The assay used a multiplexed sandwich immunoassay with dry capture anddetection reagents prepared as in Example 5. The capture antibodies wereanti-Bacillus subtilis var. Niger (BG), anti-MS2 phage, anti-FL,anti-DNP, and mouse IgG as a negative control. The dry detectionantibody pill included labeled anti-BG, anti-MS2, and anti-ovalbumin(Ova) (for detecting FL-Ova and DNP-Ova).

After the plates were prepared, the desiccant wells were filled withroughly 50 nag to 200 mg of silica gel or DRIERITE (calcium sulfate)desiccants and the plates were sealed with an aluminum foil heat seal.After sealing the foil seal to the plate top, the notches in the wellsprovided conduits between the sets of assay and desiccant wells. Someplates were prepared with no desiccant in the desiccant wells forcomparison. The plates were kept at 4° C. under dry conditions forseveral days to allow the dry reagents to fully dry. The plates werethen exposed to elevated temperature and humidity for several days priorto using them to carry out measurements of the target organisms (usingthe assay protocol of Example 5).

FIG. 10 provides signals for samples containing defined amounts of thetarget analytes and compares the signals from plates with silicadesiccant, calcium sulfate desiccant and no desiccant after exposure to60% humidity at 30° C. for 7 days. Signals are provided as a percentageof the signal obtained from a plate that was prepared at the same timeas the others but that had been kept dry a 4° C. for the 7 day period.The results indicate that the desiccant wells were very effective atimproving the stability of the dry reagents to heat and humidity.

Patents, patent applications, and publications cited in this disclosureare incorporated by reference in their entirety.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe claims.

A claim which recites “comprising” allows the inclusion of otherelements to be within the scope of the claim; the invention is alsodescribed by such claims reciting the transitional phrases “consistingessentially of” (i.e., allowing the inclusion of other elements to bewithin the scope of the claim if they do not materially affect operationof the invention) or “consisting of” (i.e., allowing only the elementslisted in the claim other than impurities or inconsequential activitieswhich are ordinarily associated with the invention) instead of the“comprising” term. Any of these three transitions can be used to claimthe invention.

1-31. (canceled)
 32. A multi-well assay plate comprising a plate bodywith a plurality of wells defined therein comprising: (a) a plurality ofdetection wells, wherein each detection well comprises a binding surfacehaving a capture reagent immobilized thereon and (b) a plurality ofreagent reconstitution wells, wherein each reagent reconstitution wellcomprises a reconstitutable labeled detection reagent, wherein at leastone detection well and one reagent reconstitution well comprise matchedcapture and detection reagents for measuring an analyte of interest. 33.A multi-well assay plate comprising a plate body with a plurality ofwells defined therein comprising: (a) a plurality of detection wells,wherein said detection wells (i) have well floors and well walls,wherein said well walls have inner wall surfaces and outer wallsurfaces, (ii) are arranged in a regular two dimensional pattern, and(iii) comprise, on an inner surfaces of each of said detection wells, abinding surface having a capture reagent immobilized thereon array; and(b) a plurality of reagent reconstitution wells, wherein said reagentreconstitution wells (i) have well floors and well walls, wherein saidwell walls are defined by outer wall surfaces of said detection wellsand by rib elements connecting the outer wall surfaces of adjacentdetection wells and (ii) comprise, in each reagent reconstitution well,a reconstitutable dry labeled detection reagent; wherein at least onedetection well and one reagent reconstitution well comprise matchedcapture and detection reagents for measuring an analyte of interest.34-43. (canceled)
 44. A multi-well plate comprising a plate body with aplurality of wells defined therein comprising: (a) a plurality of firstreagent wells holding a reconstitutable first dry reagent and (b) aplurality of second reagent wells holding a second dry reagent, whereinsaid first and second reagents are matched reagents for conducting anassay.
 45. The multi-well assay plate of claim 44, wherein said firstreagent wells are arranged in a regular two dimensional pattern and saidfirst reagent wells have well floors and well walls, said well wallshaving inner wall surfaces and outer wall surfaces and said secondreagent wells have well floors and well walls, said well walls beingdefined by outer wall surfaces of said detection wells and by ribelements connecting the outer wall surfaces of adjacent detection wells.46-82. (canceled)